Weed species have long been a problem in cultivated fields. Although weed control can be a labor intensive operation, it has been made easier by the availability of efficient weed killing chemical herbicides. The widespread use of herbicides, along with improved crop varieties and fertilizers, has made a significant contribution to the “green revolution” in agriculture. Particularly useful herbicides are those that have a broad spectrum of herbicidal activity. Unfortunately, broad spectrum herbicides typically have a deleterious effect on crop plants exposed to the herbicide. One way to overcome this problem is to produce crop plants that are tolerant to the broad spectrum herbicide.
One example of a broad spectrum herbicide is N-phosphonomethyl-glycine, also known as glyphosate. Glyphosate has been used extensively by farmers worldwide for controlling weeds prior to crop planting, for example, in no-till farming. In addition, glyphosate is an efficient means to control weeds and volunteer plants between production cycles or crop rotations. Glyphosate does not carry-over in soils after use, and it is widely considered to be one of the most environmentally safe and broadly effective chemical herbicides available for use in agriculture.
Glyphosate kills plants by inhibiting the shikimic acid pathway. This pathway leads to the biosynthesis of aromatic compounds, including amino acids, vitamins, and plant hormones. Glyphosate blocks the condensation of phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by binding to and inhibiting activity of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase, commonly referred to as “EPSP synthase,” and “EPSPS.”
Unfortunately, no crop plants are known that are naturally tolerant to glyphosate, and, therefore, the utility of this herbicide for weed control in cultivated crops has been limited. One method to produce glyphosate-tolerant crop plants is to introduce a gene encoding a heterologous glyphosate-tolerant form of an EPSPS gene into the crop plant using the techniques of genetic engineering. Using chemical mutagenesis, glyphosate tolerant forms of EPSPS have been produced in bacteria, and the heterologous genes were introduced into plants to produce glyphosate-tolerant plants. See, e.g., Comai et al. (1983) Science 221:370-71. The heterologous EPSPS genes may be overexpressed in the crop plants to obtain a desired level of tolerance.
EPSPS folds into two similar domains, each comprising three copies of a βαβαββ-folding unit (Stallings et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:5046-50). Lys-22, Arg-124, Asp-313, Arg-344, Arg-386, and Lys-411 are conserved residues of the EPSPS from E. coli (Schonbrunn et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:1376-80). Conserved residues important for EPSPS activity also include Arg-100, Asp-242, and Asp-384 (Selvapandiyan et al. (1995) FEBS Letters 374:253-6). Arg-27 binds to S3P (Shuttleworth et al. (1999) Biochemistry 38:296-302).
Variants of wild-type EPSPS have been isolated that are glyphosate-tolerant as a result of alterations in the EPSPS amino acid coding sequence (Kishore and Shah (1988) Annu. Rev. Biochem. 57:627-63; Wang et al. (2003) J. Plant Res. 116:455-60; Eschenburg et al. (2002) Planta 216:129-35). He et al. (2001) Biochim et Biophysica Acta 1568:1-6) have developed EPSPS enzymes with increased glyphosate tolerance by mutagenesis and recombination between the E. coli and Salmonella typhimurium EPSPS genes, and suggest that mutations at position 42 (T42M) and position 230 (Q230K) are likely responsible for the observed resistance. Subsequent work (He et al. (2003) Biosci. Biotech. Biochem. 67:1405-9) shows that the T42M mutation (threonine to methionine) is sufficient to improve tolerance of both the E. coli and S. typhimurium enzymes.
Currently, there are three primary classes of EPSPS that are known in the art: Class I (glyphosate sensitive); Class II (PCT International Patent Publication No. WO2006/012080 A2; Liang et al. (2009) J. Biotechnol. 144(4):330-6); and Class III (PCT International Patent Publication No. WO2007/0082269 A2; U.S. Patent Publication No. US 2010/0144530 A1).
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.